Expression of the Genes Encoding the Cytoskeletal Proteins Vimentin and Protein 4.1

Year: 1987 DOI: 10.7907/qh54-ka26
The investigations presented in this thesis represent an effort to understand the regulated expression of cytoskeletal proteins in differentiating cell systems. Vimentin is an intermediate filament protein whose expression is regulated during the differentiation of a variety of cell types. I have isolated DNA probes specific for chicken vimentin and utilized them for the study of vimentin gene regulation. The single chicken vimentin gene encodes multiple mRNAs that differ in the lengths of their 3' untranslated regions. These mRNAs are differentially expressed in a tissue-specific manner. Furthermore, vimentin mRNA increases to high levels during chicken embryonic erythropoiesis, underlying similar changes in vimentin protein accumulation.

Unlike nucleated avian erythrocytes, mammalian erythrocytes are devoid of intermediate filaments. I show that cultured murine erythroleukemia (MEL) cells repress the levels of vimentin mRNA during inducer-mediated differentiation, resulting in a subsequent loss of vimentin filaments. The expression of vimentin in these cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. To examine the molecular basis for divergent vimentin gene regulation in avian and mammalian erythropoiesis, I have studied the behavior of chicken and hamster vimentin genes introduced into MEL cells. During MEL cell differentiation, RNA encoded by transfected chicken vimentin genes significantly increases in abundance, whereas RNAs arising from either transfected hamster vimentin genes or the endogenous mouse vimentin gene are repressed. The results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences.

Protein 4.1 is an extrinsic membrane protein that facilitates the interaction of spectrin and actin in the erythroid membrane skeleton. Previous studies have shown that chicken protein 4.1 exists as a multiplet of related polypeptides that are differentially expressed during erythropoiesis. I have isolated cloned cDNA probes for chicken protein 4.1, and have found that a single protein 4.1 gene encodes multiple mRNAs by differential processing; the ratios of protein 4.1 mRNAs change during erythroid development. In vitro translation experiments demonstrate that while the expression of protein 4.1 polypeptides is specified initially at the mRNA level by RNA processing, the ultimate expression of protein 4.1 variants is further determined translationally.